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L9 Enterobacteriaceae Lecture prepared by Dr. M.

Watts Objectives students will become familiar with a range of tests designed to identify Enterobacteriaceae a range of diseases caused by these organisms Know where they fit into the classification schemes L9 Enterobacteriaceae Lecture prepared by Dr. M.Watts Objectives students will become familiar with a range of tests designed to identify Enterobacteriaceae a range of diseases caused by these organisms Know where they fit into the classification schemes Medically important bacteria: So far.. Gram + Gram-positive cocci Micrococaceae Streptococcacaea Gram-positive rods Aerobic Bacillus spp. Lactobacillus sp. Corynebacterium spp Listeria spp. Erysipelothrix rhusiopathiae Nocardia sp. Streptomyces spp. Anaerobic Actinomyces sp. Clostridium spp Now: Gram Negative Organisms Cocci (only a few) Aerobic Neisseria Moraxella Anaerobic Veillonella sp. Rods (several hundred) Aerobic Anaerobic Facultative/fermentative GNRs: Facultative/ fermentative Enterobacteriaceae Fermenting glucose and lactose Citrobacter sp. Enterobacter sp. Escherichia coli

Klebsiella pneumoniae Fermenting glucose but NOT lactose Proteus sp. Salmonella enteriditis Salmonella typhi Shigella sp. Serratia marcescens Yersinia enterocolitica Yersinia pestis Others (non-enteric) misc GNBs Pseudomonas spp. Aeromonas sp. Plesiomonas shigelloides Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus sugar fermented is hugely important: Enterobacteriaceae What are they? Where do you find them? Are they medically important? Which are most commonly encountered? How to recognise and identify

What are they? Gram negative rods grouped together on strong phenotypic grounds Most confirmed by molecular methods Currently, they are classed into 31 genera and over 100 species. Where do you find them? Primarily the bowel 1012 per gram of faeces large range of animals, both warm and cold blooded Also on some plants and in the soil . wherever you find animal faeces Prefer moist environments Where do you find them? Primarily the bowel 1012 per gram of faeces large range of animals, both warm and cold blooded Also on some plants and in the soil . wherever you find animal faeces Prefer moist environments Are they medically important? YES! Cause a range of infections Enteric: Salmonella, Shigella, E. coli. UTI: E. coli. Nosocomial: Septicaemia, Pneumonia Wound infection Have the opportunity reasonably resistant to antibiotics have the necessary virulence attributes E. coli Clinical aspects: normal flora of GIT produces vitamin K in the large intestine most commonly isolated pathogen in hospitalized patients UTI, neonatal meningitis, gastroenteritis, wound infections, pneumonia, septicaemia. LPS (endotoxin) is released when the cell dies Treating infections with antibiotics may place the patient in severe shock some strains have acquired additional genetic information from plasmids, transposons, and phages which allows them to be pathogenic E.coli: a highly variable organism 5 classes cause diarrhoeal diseases ETEC (Enterotoxigenic) EPEC (Enteropathogenic)

EIEC (Enteroinvasive) EHEC 0157 (Enterohaemorrhagic) EaggEC EHEC 0157(Enterohaemorrhagic)

How can variants of the same organism be so different? Salmonella http://video.google.com/videoplay?docid=6673841890662551798 http://video.google.com/videosearch?q=whale&emb=0#emb=0&q=beached%20whale%20new% 20zealand&src=2 How can variants of the same organism be so different? Salmonella http://video.google.com/videoplay?docid=6673841890662551798 http://video.google.com/videosearch?q=whale&emb=0#emb=0&q=beached%20whale%20new% 20zealand&src=2 Salmonella nomenclature controversial original taxonomy based on clinical considerations, e.g., Salmonella typhi, Salmonella cholerae-suis, Salmonella abortus-ovis, Now known that all Salmonella serovars form a single species (Salmonella enterica ) composed of seven subgroups Subgroup 1 contains most common serotypes typhi cholerae-suis paratyphi gallinarium pullorum Subgroup 2 : salamae Subgroup3a: arizonae Subgroup3b: diarizonae Subgroup 4: houtenae Subgroup 5: bongori Subgroup 6: indica Eg Salmonella enterica serovars (e.g., Enteritidis, Typhi, Typhimurium) Ie Salmonella ser. Typhimurium (not italicized) Some books: S. Typhimurium S. Typhi Why the problem? three kinds of major antigens Somatic (O) or Cell Wall Antigens 67 used for serological identification. Surface (capsular) Antigens Vi antigen well known Flagellar (H) Antigens Antiflagellar antibodies can immobilize bacteria with corresponding H antigens. So many surface antigens Clinical Aspects: two distinct diseases

enteric fever (typhoid), bacterial invasion of the bloodstream (S. typhi) acute gastroenteritis foodborn Salmonella: (S.typhimurium, S.enteritidis) chicken and eggs reservoir. implicated in more than 50,000 cases of bacterial food poisoning in the United States every year S. typhi only carried by humans Role of carriers About 5% of patients clinically cured from typhoid remain carriers for months or even years. Antibiotics are usually ineffective on Salmonella carriage because the site of carriage (gall bladder) may not allow penetration by the antibiotic. Outbreak of salmonella http://video.google.com/videoplay?docid=623 8435932617357831&q=&hl=en

Shigella Severe diarrhea accompanied by fever and also invasive reservoir human only small inoculum (10 to 200 organisms) is sufficient to cause infection. Shiga toxin A:B, enters cell disrupts protein synthesis Outbreaks in daycare, nursing homes spread Four f s faeces food flies fingers four species Serotype A-S. dysenteriae Serotype B-S. flexneri Serotype C-S. boydii (rare) Serotype D-S. sonnei (most common) KLEBSIELLA Klebsiella pneumoniae Common cause of nosocomial pneumonia and UTI(second only to E. coli) Produces a heat-stable enterotoxin contain resistance plasmids (R-plasmids) can be transferred to other enteric bacteria (not necessarily of the same species) Shigella Severe diarrhea accompanied by fever and also invasive reservoir human only small inoculum (10 to 200 organisms) is sufficient to cause infection. Shiga toxin A:B, enters cell disrupts protein synthesis Outbreaks in daycare, nursing homes spread Four f s faeces food flies fingers four species Serotype A-S. dysenteriae Serotype B-S. flexneri Serotype C-S. boydii (rare) Serotype D-S. sonnei (most common) KLEBSIELLA Klebsiella pneumoniae Common cause of nosocomial pneumonia and UTI(second only to E. coli) Produces a heat-stable enterotoxin contain resistance plasmids (R-plasmids) can be transferred to

other enteric bacteria (not necessarily of the same species) P. mirabilis Proteus: urinary pathogens Can form stones caused by infection of the urine with ureasplitting bacteria. Kidney Staghorn calculus Yersinia pestis Yersinia pestis rodent pathogen, The flea draws viable Y. pestis organisms into it s intestinal tract. These organisms multiply in the flea. humans an accidental host Los Angeles Times

Others . NF gut can all cause opportunistic infections EDWARDSIELLA E. tarda known to cause gastroenteritis and wound infections CITROBACTER C. freundii is suspected to cause diarrhea C. diversus has been linked to a few cases of meningitis in newborns. ENTEROBACTER E. aerogenes and E. cloacae are sometimes associated with UT & RT infections SERRATIA Serratia marcescens is considered a harmful human pathogen known to cause urinary tract infections, wound infections, and pneumonia. also have many antibiotic resistance properties Identification Uses biochemistry of bacteria Think about what they do and where they live Others . NF gut can all cause opportunistic infections EDWARDSIELLA E. tarda known to cause gastroenteritis and wound infections CITROBACTER C. freundii is suspected to cause diarrhea C. diversus has been linked to a few cases of meningitis in newborns. ENTEROBACTER E. aerogenes and E. cloacae are sometimes associated with UT & RT infections SERRATIA Serratia marcescens is considered a harmful human pathogen known to cause urinary tract infections, wound infections, and pneumonia. also have many antibiotic resistance properties Identification Uses biochemistry of bacteria Think about what they do and where they live 12 most common genera Often collectively called coliforms . Escherichia coli: type species ESCHERICHIA SHIGELLA EDWARDSIELLA SALMONELLA CITROBACTER KLEBSIELLA ENTEROBACTER SERRATIA PROTEUS MORGANELLA PROVIDENCIA YERSINIA Which tests are most useful? Diagnostic microbiology Most useful tests for quick ID Empirical: Ist stage tests Identification:

GNR E.coli, a vibrio & a pseudomonad Which is which?? OF test: most are fermenters Which is fermenter? Which is oxider? A B

Next? Have GNR fermentative : ???? how to differentiate Enterobacteriacea from the others Oxidase neg: enterobacteriacea Oxidase pos: = the others Vibrios Aeromonads Pseudomonads (aerobic) Will do these next lecture The OXIDASE test Nitrate reduction +ve for Enterobacteriaceae Detects whether bacteria uses nitrate (NO3) as electron acceptor (ie) Nitrate reduced to nitrite (or other compounds) via nitrate reductase NO3 ----> NO2 ----> NH3 or N2 Grow organism in nitrate broth: test for end products reagents: a-naphthylamine and sulfanilic acid Formation of red color after addition of the Next? Have GNR fermentative : ???? how to differentiate Enterobacteriacea from the others Oxidase neg: enterobacteriacea Oxidase pos: = the others Vibrios Aeromonads Pseudomonads (aerobic) Will do these next lecture The OXIDASE test Nitrate reduction +ve for Enterobacteriaceae Detects whether bacteria uses nitrate (NO3) as electron acceptor (ie) Nitrate reduced to nitrite (or other compounds) via nitrate reductase NO3 ----> NO2 ----> NH3 or N2 Grow organism in nitrate broth: test for end products reagents: a-naphthylamine and sulfanilic acid Formation of red color after addition of the Nitrate reduction Which are +ve? Another example: Following incubation and addition of nitrate reagents: 3 4 Which are positive?? Now which are positive? (Following addition of Zn dust) What about 1? differentiating pathogens from non-pathogens: ability to ferment lactose

0% 95% 0 -1% 5% 0 1% 2% 98% 0% 98% 2% Citrobacter freundii (50%)

Commonly used lactose containing selective and differential media MacConkey XLD EMB KIA TSI MacConkey Agar selective and differential contains lactose, bile salts, neutral red (and crystal violet). selective media Gram-positive organisms are inhibited by the bile salts and the crystal violet. Differential When bacteria ferment lactose and produce enough acid products to reduce the pH below 6.8, the neutral red turns from colorless to red. Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermenters are colorless. Commonly used lactose containing selective and differential media MacConkey XLD EMB KIA TSI MacConkey Agar selective and differential contains lactose, bile salts, neutral red (and crystal violet). selective media Gram-positive organisms are inhibited by the bile salts and the crystal violet. Differential When bacteria ferment lactose and produce enough acid products to reduce the pH below 6.8, the neutral red turns from colorless to red. Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermenters are colorless. Reactions on Mac A B C D Have a guess? XLD selective and differential medium designed for the isolation of Gram-negative enteric pathogens from clinical specimens. contains xylose, lysine, sodium desoxycholate, sodium thiosulfate and ferric ammonium citrate.

Principle: Differentiation of Shigella and Salmonella from nonpathogenic bacteria is accomplished by three reactions: 1) xylose fermentation, 2) lysine decarboxylation, and 3) hydrogen sulfide production. Xylose: enterics (except Shigella) ferment xylose rapidly. Salmonella rapidly exhaust xylose and decarboxylate lysine, and revert to alkaline conditions (simulates the Shigella reaction). Lactose and sucrose, added in excess, prevent lactose fermenters from similarly reverting. The production of hydrogen sulfide under alkaline conditions results in the formation of colonies with black centers, whereas, under acidic conditions, this black precipitation is inhibited

E. coli on XLD agar: lactose and sucrose fermented, lysine and H2S negative. Shigella on XLD agar: non-fermentive, no H2S Salmonella on XLD agar: lysine positive, xylose fermented, positive H2S (colonies with black centers) Proteus on XLD agar: swarming inhibited, xylose only fermented, lysine negative. Reactions on XLD E. coli on XLD agar: lactose and sucrose fermented, lysine and H2S negative. Shigella on XLD agar: non-fermentive, no H2S Salmonella on XLD agar: lysine positive, xylose fermented, positive H2S (colonies with black centers) Proteus on XLD agar: swarming inhibited, xylose only fermented, lysine negative. Reactions on XLD EMB: Peptones 10.0; di-potassium hydrogen phosphate 2.0; lactose 5.0; sucrose 5.0; eosin Y; methylene blue 0.07; agar-agar 13.5. KIA formula Meat extract..................................................................... ......3,00 Yeast extract.................................................................... ......3,00 Peptone.......................................................................... .......20,00 Lactose.......................................................................... ........10,00 Sodium chloride.................................................................. ... 5,00 Dextrose......................................................................... ....... 1,00 Ammonium Ferrous citrate......................................................0,5 0 Sodium thiosulfate............................................................... .... 0,50 Phenol red....................................................................... ......0,03 Agar............................................................................. ..........15,00 Final pH 7,4 0,2 1. Fermentation of glucose, lactose 2. Production of gas during fermentation 3. Production of H2S from the sulfur source Phenol red is used as PH indicator: yellow in acid, red in alkaline KIA: allows for determination

of: KIA interpretation Lactose glucose l i l t t i t i/ nonfermenters such as Pseudomonas and others K/K Salmonella, Edwardsiella, Shigella K/A E. coli, Yersinia, Aeromonas, Vibrio A/A SOME COMMON SUGAR REACTIONS IN KIA THIS MEDIUM IS RUN ON GRAM -RODS ONLY! Why include glucose?

KIA interpretation If the butt of tube is yellow (A): glucose fermenter If the slant of tube is yellow (A): lactose fermenter If the slant is red (K): non-lactose fermenter Black ppt: H2S prodn Gas: gaps in agar A/A, K/A and K/K Could you get A/K?????? (If the butt of tube is red (K): non-glucose fermenter) Carbohydrate fermentation Single CHO Test Result 1.Control Negative 2. S. aureus ? 3. P. vulgaris ?? 4. P.aeruginosa 5. E. coli ?? +ve Acid prodn: color change from red to yellow . KIA interpretation If the butt of tube is yellow (A): glucose fermenter If the slant of tube is yellow (A): lactose fermenter If the slant is red (K): non-lactose fermenter Black ppt: H2S prodn Gas: gaps in agar A/A, K/A and K/K Could you get A/K?????? (If the butt of tube is red (K): non-glucose fermenter) Carbohydrate fermentation Single CHO Test Result 1.Control Negative 2. S. aureus ? 3. P. vulgaris ?? 4. P.aeruginosa 5. E. coli ?? +ve Acid prodn: color change from red to yellow . Amino acid decarboxylation Decarboxylation only takes place in an acid environment & needs to be anaerobic Net reaction is alkaline How can you be sure decarboxylation has occurred? 2 tubes inoculated: one without amino acid but both with glucose (for .? sterile mineral oil overlay Tube without aa is control (goes yellow) Typical reactions: which set is +ve? Motility test A non-motile organism will have a clearly defined edge as it grows on the stab line

Motile organisms will be turbid throughout the tube or have fuzzy, diffuse growth at the edges. Some organisms are so motile that the entire tube becomes very turbid (cloudy). Indole-methyl red-VP-citrate IMViC tests (4 tests) used to differentiate Enterobacter and Klebsiella from E.coli three sets of media are inoculated: indole test (tryptone broth) MR-VP broth, Simmons citrate medium

The Indole Test tests the ability of organism to split indole from tryptophan (ie have tryptophanase) Indole reagents: Kovac's reagent: Pdimethylaminobenzaldehyde in alcohol Positive reaction: formation of red color at the interface of the broth and reagent tryptone broth E.coli K.pneumoniae MR-VP All enterics oxidize glucose for energy end products vary depending on bacterial enzymes MR and VP tests are used to determine what end products result MR-VP media buffered-dextrose peptone broth tests are read from a single inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes. The Indole Test tests the ability of organism to split indole from tryptophan (ie have tryptophanase) Indole reagents: Kovac's reagent: Pdimethylaminobenzaldehyde in alcohol Positive reaction: formation of red color at the interface of the broth and reagent tryptone broth E.coli K.pneumoniae MR-VP All enterics oxidize glucose for energy end products vary depending on bacterial enzymes MR and VP tests are used to determine what end products result MR-VP media buffered-dextrose peptone broth tests are read from a single inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes. Methyl red: It tests the ability of organism to produce and maintain

strong, mixed acid from buffered-dextrose peptone broth E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test) Vogues Proskauer Klebsiella and Enterobacter produce more neutral products from glucose (acetoin) pH rises above 6.2. The reagents: alpha-napthol and potassium hydroxide. If acetoin is present reagents turn a pink-burgundy color (a positive VP test). color may take 20 to 30 minutes to develop. The Citrate Test: Citrate use alkalinises the medium Simmon's medium Typical Composition (g/liter) Ammonium dihydrogen phosphate 1.0; di-potassium hydrogen phosphate 1.0; sodium chloride 5.0; sodium citrate 2.0; sole C and energy source magnesium sulfate 0.2; bromothymol blue 0.08; pH of 6.9: agar-agar 13.0. yellow at acidic pH's (around 6), and blue at more alkaline pH's (around 7.6). positive citrate: blue Enterobacter and Klebsiella + E.coli Why does citrate use alkalinise the medium?? Bacteria that use citrate also utilize the ammonium salt as a nitrogen source and create ammonia as a result.

IMViC Interpretation E.coli gives ++Enterobacter and Klebsiella give the reverse: --++ ONPG test Principle Lactose utilization requires 2 main enzymes, permease and b-galactosidase Enzymes ONLY induced in the presence of the lactose substrate (inducer) Need high lactose medium Regular LFs produce both permease enzyme and bgalactosidase True NLFs lack both of these enzymes Late LFs produce b-galactosidase but lack permease (eg Shigella sonnei) ONPG: (Ortho-nitrophenyl-b-D-galactopyranoside ) Artificial substrate turns yellow in the presence of beta-galactosidase. http://vcell.ndsu.nodak.edu/animations/lacOperon/index.htm IMViC Interpretation E.coli gives ++Enterobacter and Klebsiella give the reverse: --++ ONPG test Principle Lactose utilization requires 2 main enzymes, permease and b-galactosidase Enzymes ONLY induced in the presence of the lactose substrate (inducer) Need high lactose medium Regular LFs produce both permease enzyme and bgalactosidase True NLFs lack both of these enzymes Late LFs produce b-galactosidase but lack permease (eg Shigella sonnei) ONPG: (Ortho-nitrophenyl-b-D-galactopyranoside ) Artificial substrate turns yellow in the presence of beta-galactosidase. http://vcell.ndsu.nodak.edu/animations/lacOperon/index.htm ONPG test: detects the presence of galactosidase organism needs to be growing in/on any medium with lactose (to induce the production of galactosidase) Pipette 0.5ml of the saline into a sterile tube. Inoculate with the bacterium and add the ONPG disc in a sterile manner (forceps dipped in alcohol and flamed) to the tube. Incubate at 37C for 4 hours. INTERPRETATION: any shade of yellow + for galactosidase enzyme. Precautions A heavy inoculum is necessary to obtain a high concentration of enzyme. Urease test Some bacteria

produce urease: Urease splits urea into ammonia and carbon dioxide Organisms that produce urease will turn hot pink due to ammonia prodn Urease +ve Proteus SERRATIA characteristic red pigment.

Summary: Recognition and Identification Is it a coliform or another type of GNB? Facultative, growth on Mac: then do oxidase (-) [OF (F) and NO3 reduction tests not usually needed] Which coliform is it? Is it a major pathogen (salmonella or shigella or E.coli 0157?) Requires a battery (10-20) of biochemical tests Usually commercially packaged Many of these are broadly discriminatory. A range of CHO s added for more fine discrimination. Characteristics shared by all Enterobacteriaceae Gram negative rods oxidase negative All can ferment glucose Facultative anaerobes reduce nitrate to nitrite Summary: Recognition and Identification Is it a coliform or another type of GNB? Facultative, growth on Mac: then do oxidase (-) [OF (F) and NO3 reduction tests not usually needed] Which coliform is it? Is it a major pathogen (salmonella or shigella or E.coli 0157?) Requires a battery (10-20) of biochemical tests Usually commercially packaged Many of these are broadly discriminatory. A range of CHO s added for more fine discrimination. Characteristics shared by all Enterobacteriaceae Gram negative rods oxidase negative All can ferment glucose Facultative anaerobes reduce nitrate to nitrite Then more tests to speciate Lactose fermentation CHO fermentation Decarboxylase reactions H2S production IMViC reactions: I: indole M: methyl red V: Voges-Proskauer C: citrate And others BUT consider: Biochemical variability from tables ONPG LDC CIT ARA IND C.freundii 95 -62 + 6 K. pneumoniae + + + + E. coli 95 77 -83 94 Salmonella spp. v v v v -

Pr. mirabilis --57 -Probability and identification API strips are therefore very useful!!!!! ONPG Sorbitol Indole Result in your test + + + E. coli 95 95 98 Shigella 5 30 50 Could be either E.coli or Shigella, but E. coli more probable. More tests in your panel = better probability.

The use of codes and code books Do tests in set order group the results in sets of three. Assign 0 for a (-) and 1, 2 or 4 for a (+). ---= 0, + --=1, -+ -= 2, + + -= 3 --+ = 4, + -+ = 5, -+ + = 6, + + + =7 ( note that if 1, 2, 3 then + + -=3 and --+ = 3) 514 is unique to those results. 415 400001401 +----++-+ IND H2S CIT LDC ARA XYL VPMR MOT API profile result sheet Spot the deliberate mistake!!!!!! The use of codes and code books Do tests in set order group the results in sets of three. Assign 0 for a (-) and 1, 2 or 4 for a (+). ---= 0, + --=1, -+ -= 2, + + -= 3 --+ = 4, + -+ = 5, -+ + = 6, + + + =7 ( note that if 1, 2, 3 then + + -=3 and --+ = 3) 514 is unique to those results. 415 400001401 +----++-+ IND H2S CIT LDC ARA XYL VPMR MOT API profile result sheet Spot the deliberate mistake!!!!!! Mechanics of identification Do the 10 or 20 tests. + --+ + -+ + + --+ + + --+ + Number? 137436 Now compare your results with a table of expected results for over 100 different organisms. This could be a table of 30 x 100! Automation Many systems now available. organism in diluent. aspirates into black box Distributes inoculum into wells with dehydrated substrates and incubates. Instrument reads results with spectro. Determines + or -, and compares with database. Prints out name of organism. Major ID tests GNB After growth on Mac, do oxidase test. If oxidase negative, do biochemical tests. Carbohydrate fermentation KIA agar/TSI IMViC Urease Decarboxylase reactions ONPG H2S

Motility Compose a flow charts from major tests

Enterobacteriaceae flow chart End of lecture Review questions Enterobacteriaceae flow chart End of lecture Review questions